Thomas R. Howdieshell

The Importance of Engraftment in Flap Revascularization: Confirmation by Laser Speckle Perfusion Imaging

BACKGROUND The delivery of proangiogenic agents in clinical trials of wound healing has produced equivocal results, the lack of real-time assessment of vascular growth is a major weakness in monitoring the efficacy of therapeutic angiogenesis, and surgical solutions fall short in addressing the deficiency in microvascular blood supply to ischemic wounds. Therefore, elucidation of the mechanisms involved in ischemia-induced blood vessel growth has potential diagnostic and therapeutic implications in wound healing. MATERIALS AND METHODS Three surgical models of wound ischemia, a cranial-based myocutaneous flap, an identical flap with underlying silicone sheeting to prevent engraftment, and a complete incisional flap without circulation were created on C57BL6 transgenic mice. Laser speckle contrast imaging was utilized to study the pattern of ischemia and return of revascularization. Simultaneous analysis of wound histology and microvascular density provided correlation of wound perfusion and morphology. RESULTS Creation of the peninsular-shaped flap produced a gradient of ischemia. Laser speckle contrast imaging accurately predicted the spatial and temporal pattern of ischemia, the return of functional revascularization, and the importance of engraftment in distal flap perfusion and survival. Histologic analysis demonstrated engraftment resulted in flap revascularization by new blood vessel growth from the recipient bed and dilatation of pre-existing flap vasculature. CONCLUSIONS Further research utilizing this model of graded wound ischemia and the technology of laser speckle perfusion imaging will allow monitoring of the real-time restitution of blood flow for correlation with molecular biomarkers of revascularization in an attempt to gain further understanding of wound microvascular biology.

Reliability of in vivo mixed venous oximetry during experimental hypertriglyceridemia

Background and MethodsThe spectral absorbance of iv lipid emulsion produces interference in the in vitro spectrophotometric measurement of hemoglobin saturation. Therefore, we investigated in vivo mixed venous oximetry during lipid emulsion infusions. Boluses of lipid emulsion, increasing by 0.1-g/kg increments, were infused every 30 mins into nine anesthetized dogs. After each lipid bolus, laboratory and hemodynamic data measurements were repeated, and arterial and mixed venous blood gases were analyzed on a laboratory cooximeter that was equipped for canine blood. The in vivo mixed venous oxygen saturation was continuously monitored via an indwelling, optically equipped pulmonary artery catheter. ResultsAs expected, increasing concentrations of lipid emulsion increased (r2 = .92) serum triglyceride concentration. As a result, the in vitro measurements of percentage methemoglobin increased artifactually, reducing in vitro measurements of arterial (r2 = .74) and mixed venous (r2 = .44) oxygen saturations. The in vivo mixed venous oxygen saturation, however, remained constant during hypertriglyceridemia (r8 <). Conclusions: In vivomixed venous oximetry remained constant during hypertriglyceridemia, while in vitro measurements of arterial andmixed venous saturations were artifactually reduced.

The role of monocyte subsets in myocutaneous revascularization

BACKGROUND The controlled recruitment of monocytes from the circulation to the site of injury and their differentiation into tissue macrophages are critical events in the reconstitution of tissue integrity. Subsets of monocytes/macrophages have been implicated in the pathogenesis of atherosclerosis and tumor vascularity; however, the significance of monocyte heterogeneity in physiologic neovascularization is just emerging. MATERIALS AND METHODS A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of wild-type mice (C57BL6) and populations of mice with genetic deletion of subset-specific chemokine ligand-receptor axes important in monocyte trafficking and function (CCL2(-/-) and CX3CR1(-/-)) (n=36 total; 12 mice per group, nine with flap and three unoperated controls). Planimetric analysis of digital photographic images was utilized to determine flap surface viability in wild-type and knockout mice. Real-time myocutaneous flap perfusion and functional revascularization was determined by laser speckle contrast imaging. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Macrophage quantification and localization in flap tissues was determined by F4/80 gene and protein expression. Quantitative reverse transcription-polymerase chain reaction was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression. RESULTS Myocutaneous flaps created on wild type and CX3CR1(-/-) mice were engrafted to the recipient site, resulting in viability. In contrast, distal full thickness cutaneous necrosis and resultant flap dehiscence was evident by d 10 in CCL2(-/-) mice. Over 10 d, laser speckle contrast imaging documented immediate graded flap ischemia in all three groups of mice, functional flap revascularization in wild type and CX3CR1(-/-) mice, and lack of distal flap reperfusion in CCL2(-/-) mice. Immunostaining of serial histologic specimens confirmed marked increases in microvascular density and number of macrophages in wild type mice, intermediate increases in CX3CR1(-/-) mice, and no significant change in vessel count or macrophage quantity in CCL2(-/-) mice over the study interval. Finally, quantitative reverse transcriptase polymerase chain reaction demonstrated that the loss of function of chemokine ligand and receptor genes influenced the transcription of local genes involved in monocyte chemotaxis and wound angiogenesis. CONCLUSIONS In a graded-ischemia wound healing model, monocyte recruitment was severely impaired in CCL2(-/-) mice, resulting in failure of flap revascularization and concomitant cutaneous necrosis. Analysis of CX3CR1-deficient mice revealed adequate monocyte recruitment and revascularization for flap survival; however, the myeloid cell response and magnitude of neovascularization were dampened compared with wild type mice.

Pattern recognition receptor gene expression in ischemia–induced flap revascularization

BACKGROUND The innate immune system is the major contributor to acute inflammation induced by microbial infection or tissue damage. Germline-encoded pattern recognition receptors (PRRs) are responsible for sensing the presence of micro-organisms and endogenous molecules released from damaged cells. We performed microarray analyses on ischemic wound tissue to investigate the temporal relationship between PRR gene expression, wound perfusion, and flap revascularization. METHODS A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of C57BL6 mice (n = 25 total; n = 20 with flap). Laser speckle contrast imaging was utilized to study the pattern of flap ischemia and return of functional revascularization. Flap microvascular density was determined by image analysis of CD-31-immunostained sections. Total RNA was isolated from homogenized flap tissue and was converted to cDNA (RT), which was hybridized to a microarray of pathway-focused genes. Microarray results were validated with quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS Laser speckle contrast imaging predicted the spatial and temporal pattern of ischemia and functional revascularization. Histologic analysis demonstrated early leukocyte infiltration and later engraftment, resulting in flap revascularization by new blood vessel growth from the recipient bed and dilatation of preexisting proximal flap vasculature. qRT-PCR demonstrated significant early gene expression of select PRRs, cytokines, chemokines, and growth factors, peaking by 48 hours, and returning toward baseline but remaining elevated at 10 days. CONCLUSION Surgical and ischemic tissue injury resulted in the early gene expression of select PRRs, which may bind with endogenous molecules released from ischemic or necrotic cells, leading to transcription of genes involved in wound inflammation and angiogenesis.

Effects of free glycerol contained in intravenous fat emulsion on plasma triglyceride determination.

BACKGROUND Plasma triglyceride analyses to evaluate the clearance of IV fat emulsion are necessary in patients receiving parenteral nutrition. Enzymatic kits for triglyceride analysis measure the glycerol that is hydrolyzed from triglyceride. This study investigates the effect of fat emulsion free glycerol on plasma triglyceride determination. METHODS Venous blood from fasting volunteers (n = 10) was drawn into tubes containing EDTA and was centrifuged to separate the plasma. An IV fat emulsion containing 2.25% glycerol was added to plasma samples to create serial dilutions ranging from 0.049% to 0.245% final lipid concentration. Total triglyceride and free glycerol concentrations were determined in each dilution and control sample. The free glycerol concentration was subtracted from the total triglyceride concentration to yield a true triglyceride value. RESULTS Increasing concentrations of fat emulsion added to donor plasma produced increases in total triglyceride and free glycerol concentrations. The increase in free glycerol concentration produced significant concentration-related differences between total and true triglyceride concentrations. CONCLUSIONS The total triglyceride level overestimated the true triglyceride concentration due to fat emulsion free glycerol. To assure reliable triglyceride results in patients receiving fat emulsion, blanking for free glycerol should be considered.

The omentum is a site of stromal cell–derived factor 1α production and reservoir for CXC chemokine receptor 4–positive cell recruitment

BACKGROUND The mechanism of the omental response to injury remains poorly defined. This study investigates the omental reaction to a foreign body, examining the role of a chemokine ligand/receptor pair known to play a crucial role in angiogenesis and wound healing. METHODS A ventral hernia, surgically created in the abdominal wall of 6 swine, was repaired with silicone sheeting to activate the omentum. Omental thickness was determined by ultrasonography. Serial stromal cell-derived factor 1alpha (SDF-1alpha) concentrations were measured in blood, wound, and peritoneal fluids by enzyme-linked immunosorbent assay. RESULTS During the 14-day study period, serial ultrasonography showed a 20-fold increase in omental thickness, and enzyme-linked immunosorbent assay revealed a 4-fold increase in SDF-1alpha concentration in local wound fluid. Omental vessel count and vascular surface area were 8- to 10-fold higher in reactive omentum. Immunohistochemistry showed nearly complete replacement of control omental fat with CXC chemokine receptor 4 (CXCR4)-positive cells by day 14. CONCLUSIONS Activated omentum, important in the SDF-1alpha/CXCR4 axis, may serve as an intraperitoneal reservoir for recruitment of circulating bone marrow-derived cells vital to healing.

Effects of intraluminal and extracorporeal inferior vena caval bypass on canine hemodynamics

OBJECTIVE To compare inferior vena cava-right atrial extracorporeal bypass with intraluminal atriocaval shunting during hepatic vascular isolation. DESIGN Prospective, randomized, controlled animal study. SETTING University research laboratory. SUBJECTS Adult mongrel dogs (n = 5) weighing 20 to 27 kg. INTERVENTIONS Anesthetized dogs underwent laparotomy and sternotomy for vascular isolation. For atriocaval shunting, 20- and 24-Fr intraluminal shunts were inserted into the inferior vena cava via right atriotomy. For extracorporeal bypass, each animal underwent inferior vena cava, portal vein, and right atrial cannulation for venovenous bypass, utilizing a centrifugal pump. Hemodynamic data were recorded at baseline and at intervals after caval occlusion, Pringle maneuver, and caval occlusion with Pringle maneuver. MEASUREMENTS AND MAIN RESULTS Isolated Pringle maneuver and caval occlusion with Pringle maneuver produced significant reductions in mean arterial pressure (MAP) and cardiac output, irrespective of pulmonary artery occlusion pressure. Extracorporeal bypass, including both caval and portal venous return, produced significant increases in MAP and cardiac output during caval occlusion with Pringle maneuver, while atriocaval shunting and extracorporeal bypass without portal venous return did not improve MAP or cardiac output. CONCLUSION Venovenous extracorporeal bypass with portal return, acting as a right ventricular assist device, is superior to intraluminal atriocaval shunting in maintaining hemodynamic stability during hepatic vascular isolation.

Investigation of Open Abdomen Visceral Skin Graft Revascularization and Separation from Peritoneal Contents

Background: Despite increasing survival following damage control laparotomy and open abdomen technique, little is known about the biology of visceral skin graft revascularization and separation from peritoneal contents. Methods: Following laparotomy for trauma, patients with visceral edema preventing fascial closure underwent Vicryl mesh closure followed by visceral split-thickness skin grafting and readmission graft excision and abdominal wall reconstruction. Utilizing laser speckle contrast imaging, immunochemical staining of histologic sections, and RT-PCR array technology, we examined the revascularization, microvascular anatomy, morphology, and change in gene expression of visceral skin grafts. Results: Ten patients ranging in age from 25 to 46 years underwent visceral grafting for cutaneous coverage of an open abdomen. Skin graft perfusion peaked at a mean of 350 PU by post-operative day 14 synchronous with closure of meshed interstices, and remained constant until excision. Time to graft excision ranged from 6 to 18 months. CD-31 immunostaining documented a significant (p = 0.04) increase in vascular surface area in excised grafts compared to control skin. Trichrome staining revealed an 8-fold increase in excised graft thickness. Mesothelial cells were identified within the dermal matrix of excised grafts. RT-PCR demonstrated significant up-regulation of genes involved in matrix structure and remodeling, cytoskeleton regulation, and WNT signaling; and down-regulation of genes involved in inflammation and matrix proteolysis in excised grafts compared to control skin. Conclusion: Our data document early visceral skin graft perfusion and a plateau in revascularization. Histology reveals a robust dermal matrix populated by fibroblasts and mesothelial cells within a complex supporting vascular network. Genetic analysis of excised grafts reveals growth factor, collagen, and matrix remodeling gene expression.

Myocutaneous revascularization following graded ischemia in lean and obese mice

Background Murine models of diabetes and obesity have provided insight into the pathogenesis of impaired epithelialization of excisional skin wounds. However, knowledge of postischemic myocutaneous revascularization in these models is limited. Materials and methods A myocutaneous flap was created on the dorsum of wild type (C57BL/6), genetically obese and diabetic (ob/ob, db/db), complementary heterozygous (ob+/ob−, db+/db−), and diet-induced obese (DIO) mice (n=48 total; five operative mice per strain and three unoperated mice per strain as controls). Flap perfusion was documented by laser speckle contrast imaging. Local gene expression in control and postoperative flap tissue specimens was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Image analysis of immunochemically stained histologic sections confirmed microvascular density and macrophage presence. Results Day 10 planimetric analysis revealed mean flap surface area necrosis values of 10.8%, 12.9%, 9.9%, 0.4%, 1.4%, and 23.0% for wild type, db+/db−, ob+/ob−, db/db, ob/ob, and DIO flaps, respectively. Over 10 days, laser speckle imaging documented increased perfusion at all time points with revascularization to supranormal perfusion in db/db and ob/ob flaps. In contrast, wild type, heterozygous, and DIO flaps displayed expected graded ischemia with failure of perfusion to return to baseline values. RT-PCR demonstrated statistically significant differences in angiogenic gene expression between lean and obese mice at baseline (unoperated) and at day 10. Conclusion Unexpected increased baseline skin perfusion and augmented myocutaneous revascularization accompanied by a control proangiogenic transcriptional signature in genetically obese mice compared to DIO and lean mice are reported. In future research, laser speckle imaging has been planned to be utilized in order to correlate spatiotemporal wound reperfusion with changes in cell recruitment and gene expression to better understand the differences in wound microvascular biology in lean and obese states.

HEPARIN VERSUS CITRATE REGIONAL ANTICOAGULATION DURING AUTOTRANSFUSION IN A PORCINE INTRAABDOMINAL HEMORRHAGE MODEL

Our objective was to determine the effects of anticoagulants and blood loss on hemodynamic, hematologic, and coagulation parameters following autotransfusion in an animal model of intraabdominal hemorrhage. We performed a prospective, randomized observational animal study at an animal research laboratory at a university medical center. Eight Landrace, domestic pigs, weighing 17-23 kg, each underwent jugular venous and iliac arterial catheterization and laparotomy with retroperitoneal dissection for aortic exposure to simulate an operative environment. Following baseline laboratory and hemodynamic determinations, intra-abdominal hemorrhage was accomplished via aortotomy in three sequential 10 mL/kg blood volumes. After allowing pooling in the exposed retroperitoneum to ensure tissue contact, the shed blood was suctioned, processed, and washed in an autotransfusion device utilizing either heparin (n = 4) or acid-citrate-dextrose (n = 4) as a system anticoagulant. Prior to autologous transfusion, each pig received a 20 mL/kg intravenous bolus of 0.9 per cent saline to treat shock. The processed blood was then infused, and laboratory and hemodynamic measurements were repeated following each cycle of hemorrhage and autotransfusion. Sequential fixed volume hemorrhage resulted in significant reductions in mean arterial pressure. Despite crystalloid infusion and transfusion of processed shed blood, postresuscitation mean arterial pressure did not return to baseline values, with no difference noted between anticoagulant groups. Infusion of increasing volumes of autologous blood resulted in significant reductions in hematocrit, platelet count, fibrinogen, antithrombin III, ionized calcium, and total protein. The decrease in concentration of each variable was independent of the choice of anticoagulant with the exception of antithrombin III, with higher levels noted in animals receiving blood anticoagulated with acid-citrate-dextrose. Prothrombin time and partial thromboplastin time were unaffected by volume of autologous transfusion or choice of anticoagulant. We conclude that changes in hemodynamic, hematologic, and coagulation parameters associated with hemorrhage and autotransfusion appear related more to the volume of blood loss and the cumulative pheresis of plasma than to the choice of anticoagulant.